Marius Henkel

Background: Precision fermentation is a key technology for the future sustainable production of food proteins, bulk ingredients, and high-value recombinant products. However, process efficiency is often limited by the coupling of biomass formation and recombinant protein synthesis, leading to substantial carbon losses into non-recyclable biomass. Particularly for cost-sensitive bulk products such as food proteins, economically viable production requires highly efficient and dynamically controllable bioprocesses. RNA thermometers (RNATs) offer a promising inducer-free strategy for temperature-dependent control of recombinant protein production and dynamic bioprocess regulation, with the potential to improve carbon efficiency and product yields. Results: In this study, a fluorescent reporter protein (EmGFP) under the control of a fourU (4U) RNA thermometer was evaluated in E. coli BL21 across a temperature range of 25–40 °C. Temperature-dependent regulation enabled maximal recombinant protein production at 35 °C, resulting in a 2.6-fold higher specific productivity compared to the derepressed control. The strongest RNAT-mediated regulatory effect was observed at 35.5 °C, highlighting the relevance of precise temperature control for recombinant protein production. Evaluation of altered carbon partitioning revealed a substantially increased carbon allocation toward product formation, while carbon partitioning into biomass and CO ₂ remained largely unchanged. Furthermore, the product-per-acetate yield increased more than threefold at induction temperature, indicating controllable carbon partitioning and reveals potential for decoupling approaches of biomass formation from recombinant protein production. Conclusion: This study demonstrates the successful implementation of an RNAT-controlled recombinant expression system enabling temperature-dependent regulation of protein production in E. coli . Quantitative analysis revealed controllable carbon partitioning toward recombinant protein synthesis and defined target temperatures for future process induction and switching. In contrast to conventional inducer-based systems, RNAT-mediated regulation enables dynamic control without chemical additives, supporting more resource-efficient bioprocess operation. However, fully exploiting RNAT-based process control will require advanced model-based strategies integrating temperature and feeding regimes, particularly for cost-sensitive precision fermentation processes such as food protein production. Overall, RNATs represent promising molecular control elements for future high-efficiency precision fermentation processes.

Introduction: Despite growing interest in cultured meat, scaling its production from laboratory systems remains challenging. Its industrial translation is constrained by a lack of process-level models and validated engineering data. Methods: In this study, we developed a process model for animal cell biomass production in suspension batch culture using SuperPro Designer v12. The model describes suspension-based cell proliferation to produce unstructured biomass without microcarriers and excludes cell differentiation, tissue maturation, or structured tissue formation. It integrates media preparation, sequential cell expansion up to a 20,000 L stirred-tank reactor, downstream clarification and washing, extrusion, and supporting utilities into a single flowsheet. Unit operations were parameterized using literature-derived assumptions from mammalian suspension culture, enabling complete mass and energy balances throughout the system. Results: The baseline model assumes a maximum cell density of 5 × 10 ⁷ cells/mL, and yields ~2,000 kg of biomass per 30.7-day batch. Media preparation and sterilization, cleaning-in-place- and cooling-related utility demand, and cell expansion performance emerged as major scale-up bottlenecks. Scale-out simulation revealed disproportionally high utility and cultivation time demands relative to production output. Waste and utility streams, primarily depleted medium, cleaning-in-place effluents, and cooling demand, emerge as critical targets for sustainability assessment and valorization. Discussion: The model is not intended as a final process design but as a reference framework: it makes assumptions explicit, provides reproducible balances, and supports comparative scenario analysis. This framework enables systematic evaluation of scalable cultivated biomass production concepts, highlights technical potential and infrastructure requirements, and helps prevent misdirected investments by allowing process configurations to be assessed before industrial implementation.

Oxygen transfer is a critical design parameter in laboratory‐scale bioprocess systems used for prototyping, process development, and scale‐down studies of mammalian cell cultures, particularly when cultivating shear‐sensitive mammalian cells. In this work, we present the design and characterization of a 3D‐printed modular, membrane‐based aeration module that enables bubble‐free oxygen transfer in laboratory reference cell‐cultivation systems. The aeration module was developed as an external, small‐scale unit intended for flexible integration into laboratory bioreactors and perfusion setups. Fabricated via fused deposition modeling, the final design features a three‐chamber membrane‐stacking architecture that ensures mechanical stability, tightness, and biocompatibility, while allowing for straightforward adaptation through editable CAD files. The system was experimentally evaluated with respect to oxygen transfer performance under varying relative liquid flow rates and membrane configurations (PTFE and PVDF), each with two different pore sizes (0.22 µm and 0.45 µm). Key performance parameters of the aeration module were determined and include dissolved oxygen (DO) profiles, volumetric oxygen transfer coefficients (7.26 h ⁻¹ ), oxygen transfer rates (OTRs) (max. 61.4 mg L ⁻¹ h ⁻¹ ), and the pressure‐normalized oxygen mass transfer rate (0.87 g m ^{− 2} bar ^{− 1} h ^{− 1} ). Overall, the modular design and quantified performance provide a versatile tool for rapid iteration and evaluation of membrane‐based oxygenation strategies in early‐stage bioprocess development.

Bioreactors encompass a wide spectrum of engineering concepts, yet for decades, industrial biotechnology has seen little fundamental innovation, with large-scale production mainly confined to stirred-tank and bubble-column reactors for microbial fermentations. In pharmaceutical biotechnology, various cultivation systems for eukaryotic cells are well established, including microcarrier processes, suspension in stirred vessels, and disposable bags or wave (rocking) bioreactors. However, these systems have typically been optimized to produce specific biomolecules, such as recombinant proteins, vaccines, or monoclonal antibodies, rather than for generating cellular biomass itself. The emergence of tissue engineering for medical applications brought new technological impulses, particularly in the development of scaffold materials and the spatial organization of cells in three-dimensional constructs. This was accompanied by advances in perfusion bioreactors, enabling sustained nutrient delivery and waste removal within thick tissue cultures. Such systems were designed for relatively small, high-value applications in regenerative medicine, but they provide valuable conceptual and technical groundwork for structured cultivated meat manufacturing. In cellular agriculture, the goal is to apply and adapt established bioprocessing knowledge to a new challenge: producing animal cells at food-relevant scales and costs. Rather than high-value, low-volume pharmaceutical production, the focus shifts to high-volume, cost-efficient food production. Achieving this will require interdisciplinary collaboration between bioprocess engineers, automation specialists, tissue engineers, cell biologists, and food technologists. Graphical Abstract

Precise quantification of recombinant proteins is essential for assessing and comparing expression efficiency and optimizing production processes. Fluorescent proteins have emerged as powerful tools for real-time monitoring of gene expression and protein tracking. However, standardized and validated methods for their quantification, particularly for the widely used green fluorescent protein, remain limited. To date, no universally adopted protocol has emerged. This study presents a high-throughput method for the quantification of recombinantly produced Emerald Green Fluorescent Protein (EmGFP) based on direct fluorescence measurements of the cell suspension while quantifying and integrating potential effects of signal attenuation. The workflow uses solely standard laboratory equipment, ensuring broad accessibility and easy implementation. Moreover, in-house EmGFP standard preparation and quantification is described. The method was validated according to FDA guidelines “Analytical Procedures and Methods Validation for Drugs and Biologics,” addressing the requirements of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and recovery rate. Investigation was conducted using Escherichia coli BL21 cells expressing EmGFP, widely available sodium fluorescein as a chemical standard, commercial GFP, and an in-house EmGFP standard. A robust correlation (linear fitting, R ² 0.96) of the EmGFP concentration and relative fluorescence units (RFU) was established, enabling efficient and high-throughput fluorescence quantification using a standardized workflow in a microtiter-based format suitable for the application in comparative studies across different expression constructs, conditions, and scales. By enabling absolute quantification of fluorescent proteins, this method supports both real-time bioprocess optimization and broader applications in protein production research.

Eggs are among the most widely consumed and versatile animal-derived foods and are valued for their exceptional nutritional and functional properties. However, conventional egg production is associated with significant environmental, ethical, and health concerns, increasing the global consumer demand for sustainable animal protein. To address these challenges, recombinant egg protein production through precision fermentation has emerged as a promising alternative. Yet, this alternative production path is still in its infancy, and current efforts in research have not yet led to a widespread adoption of recombinant egg protein. This review provides an overview of the bioprocesses used to produce recombinant egg proteins, highlighting their nutritional, bio-functional, and techno-functional significance. The current state of the art of recombinant egg protein production is presented, with a comparison of different microbial expression hosts in terms of suitability and associated challenges. Only six egg proteins were reported to be expressed at laboratory scale, including ovalbumin (3.7 g/L with Escherichia coli EcN) and ovomucoid (3.2 g/L with Komagataella phaffii). The realization of large-scale production of functional egg proteins remains challenging. These challenges include posttranslational modifications, achieving functionality and cost parity to natural egg proteins, efficient resource bioconversion, and optimizing the bioprocess chain (upstream, bioproduction, and downstream processes). This requires further improvements and research to increase protein titers, space-time yields, and production rates. Nevertheless, recombinant egg protein produced via precision fermentation holds great promise as a functional food ingredient. With further advancements, this approach could contribute to global protein demand, enhance food security, and strengthen food system resilience while providing a more sustainable and ethical alternative to conventional egg production.